CN113584118A - Method for rapidly detecting infectious microbe drug sensitivity in blood - Google Patents
Method for rapidly detecting infectious microbe drug sensitivity in blood Download PDFInfo
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- CN113584118A CN113584118A CN202110843165.5A CN202110843165A CN113584118A CN 113584118 A CN113584118 A CN 113584118A CN 202110843165 A CN202110843165 A CN 202110843165A CN 113584118 A CN113584118 A CN 113584118A
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- bacteria
- target plate
- hole sites
- drug
- drug sensitive
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Abstract
The invention relates to the technical field of drug sensitivity detection, in particular to a method for rapidly detecting infectious microbe drug sensitivity in blood. The method comprises the following steps: mixing the blood positive sample with a surfactant solution, centrifuging and removing a supernatant; cleaning to obtain precipitated thalli; preparing a bacterial suspension; adding the bacterial suspension to a drug sensitive chip target plate, enabling the quantity of bacteria on each sample application position on the drug sensitive chip target plate to be lower than a mass spectrum detection threshold value, then carrying out incubation, absorbing and removing upper liquid after the incubation is finished, and carrying out mass spectrum detection on substances on the sample application positions on the drug sensitive chip target plate; and (4) interpreting the drug sensitivity result according to the mass spectrum detection result, and determining the sensitive, intermediary and drug resistance information. The invention can directly carry out the drug sensitivity test on the pretreated clinical yang blood reporting bottle without switching and purifying operations again, and compared with the prior method, the invention not only can save time, but also has relatively simple and rapid operation, no need of high manual ability, and lower requirement on the professional degree of personnel.
Description
Technical Field
The invention relates to the technical field of drug sensitivity detection, in particular to a method for rapidly detecting infectious microbe drug sensitivity in blood.
Background
In recent years, the incidence rate of bacteremia or fungemia is continuously increased, the bacteremia or fungemia is the first in various infections, the mortality rate is high (20% -50%), the harm is serious, and the survival rate of patients is reduced by 7.6% after the identification report time of pathogenic bacteria of bloodstream infection is delayed by 1 h. The rapid diagnosis and timely administration of bloodstream infections by clinicians are key factors in improving survival rates. Blood culture is the primary means of diagnosing bloodstream infections. The blood culture is to collect blood sample of patient, inoculate it into culture bottle, and culture for 24h-5d to increase bacteria, so as to find and identify pathogenic microorganism causing bacteremia or fungemia. The blood culture results have very important clinical significance for diagnosis, treatment and prognosis of infectious diseases.
At present, the main steps of the detection of blood culture positive specimens are that a disposable sterile syringe is used for extracting culture solution to a blood plate and a chocolate plate for transfer culture, simultaneously, the gram staining microscopy is carried out, and the staining result is reported as a critical value according to the staining reaction and the morphological characteristics of strains; and selecting bacterial colonies from the plate subjected to transfer culture for gram staining, selecting an identification step and a drug sensitivity test according to a staining result, and finally reporting the identification result and the drug sensitivity test to clinical results. According to the conventional steps, the fastest identification result of the blood culture positive specimen needs 18-48h, and the fastest identification result of the drug sensitive specimen needs 20-48 h.
The conventional microorganism detection method comprises morphological characteristics, physiological and biochemical reactions, serological reactions and sequencing, wherein the morphology and the biochemistry are the most common methods in clinic at present, the morphological method is to preliminarily identify species according to the growth state of microorganisms on a solid/liquid culture medium, a dyeing reaction and a microscopic observation result, the method is low in cost and high in detection speed, but has high professional requirements on operators, most of microorganism forms have no obvious species specificity, misjudgment is easy to occur, and the identification types are limited. The physiological and biochemical reaction method has a complex flow, needs to judge whether the bacteria are gram-positive bacteria or gram-negative bacteria, and then identifies the specific microorganism types according to different methods such as morphology, enzyme reaction, color development and the like. The serological reaction is to utilize antiserum of known strains to observe whether the serological reaction with the specificity of a to-be-identified strain occurs or not to identify the type of the to-be-identified strain, the technical detection speed is high, but the method is only a supplement means of other identification methods, the operation is complex, the requirement on operators is high, a special kit needs to be purchased for serological detection, the cost is high, the technical limitation degree is high, clinical strains which can be detected by serology generally can be subjected to serological detection only after the name of the strain is roughly determined, common salmonella, shigella and the like exist, and the number of applicable strains is small. The sequencing technology is used for clarifying the taxonomic relation among microbial populations from the genetic evolution perspective, does not depend on the characteristics of strains, can be used for all strains, has high identification accuracy and is the gold standard of the existing microbial identification technology. However, the sequencing technology has high detection cost and long time consumption, so that the sequencing technology is difficult to popularize in large scale in clinic at present, and the traditional microorganism detection method (form/biochemistry) is still mainly used in clinic. With the different complexity of the infected microorganisms, the traditional microorganism detection time is as long as several days and as short as several hours, and the types which can be identified are limited and the accuracy is general. Compared with the traditional method, the MALDI-TOF MS mass spectrometer has the absolute advantages of high identification speed, accuracy, low cost and the like, and becomes one of the most vital new technologies in the field of clinical microbiological examination.
At present, scholars at home and abroad try to apply the mass spectrometry technology to the direct detection of clinical samples, and remarkable progress is achieved, different pretreatment technologies, centrifugation, filtration, surfactants and the like exist for different experts of different samples, and the technology of directly detecting the clinical samples through mass spectrometry is gradually approved by clinical and microbiological laboratories, so that the time of clinical reports is greatly shortened.
However, there is a need in the medical field not only for rapid identification, but also for detection of resistance to commonly used antibiotics. Since the abuse of antibiotics in recent years causes a great increase of drug-resistant bacteria, and diseases cannot be effectively controlled and treated clinically without knowing the drug resistance of the antibiotics, the rapid identification of infectious microorganisms in blood or other body fluids and the rapid determination of the drug resistance of the microorganisms to different antibiotics are necessary, which is of great significance for clinical anti-infective treatment.
The conventional drug sensitivity detection methods are more, and mainly comprise a paper diffusion method, a trace broth dilution method (gold standard), an E-test method and a full-automatic drug sensitivity instrument method. (1) The paper diffusion method comprises uniformly coating the prepared bacterial suspension on MH agar culture medium, drying at room temperature, attaching medicated paper to the surface of the culture medium, incubating for 16-24h, judging according to the measured diameter of the inhibition zone and CLSI standard, and reporting sensitivity (S), medium (I) and resistance (R); (2) a trace broth dilution method is characterized in that a series of double dilutions are carried out on an antibacterial drug with a certain concentration and a culture solution of bacteria to be detected, and after the constant-temperature culture is carried out for 16-24h, the minimum inhibition concentration and MIC value of the drug to the bacteria to be detected are judged by observing the growth phenomenon of the bacteria in the culture solution; (3) e-test method, which is to evenly coat the prepared bacterial suspension on MH agar culture medium, dry the bacterial suspension at room temperature, tightly attach the medicated paper sheet on the surface of the culture medium, incubate for 16-24h, and directly measure the MIC value of the drug to the bacteria to be measured by observing the bacterial growth phenomenon on the MH agar culture medium; (4) a full-automatic microbial drug sensitivity detection system is an intelligent microbial drug sensitivity detection system, the principle of the system is a trace broth dilution method, the operation is relatively simple, only a certain McLee unit of bacterial suspension needs to be prepared, the bacterial suspension is inserted into a drug sensitivity card to be automatically detected, and the drug resistance result and the MIC value are checked within about 10-24 hours.
The first three methods are recommended by CLSI, wherein the broth dilution method is regarded as the gold standard, but the operation steps are complicated, the time is as long as 16-24 hours, and the detection requirement of clinical critical patients is difficult to meet. The paper diffusion method has high requirements on manual operations such as the quality of drug sensitive paper, the coating of bacteria liquid on MH agar culture medium, the measurement of bacteriostatic zones and the like, the dependence on personnel is high, the accuracy of a detection result is poor, the identification time is as long as 16-24 hours, and the detection requirement of clinical critical patients is difficult to meet. The full-automatic microbial drug sensitivity detection system is simple and convenient to operate, is relatively quick to operate, but has relatively high dependence on drug sensitivity board cards, the types of the drug sensitivity board cards adopted in the market at present are single, the range of drug concentration gradient is relatively narrow, additional detection may be required according to requirements clinically, long-term monitoring of drug resistance is not facilitated, the consumed time is relatively long, and the methods cannot perform comprehensive drug sensitivity detection on patients in time.
Disclosure of Invention
In view of this, the present invention provides a method for rapidly detecting infectious microbial susceptibility in blood. The method is simple to operate, and can greatly shorten the time for identifying blood infectious microorganisms and detecting drug sensitivity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for rapidly detecting infectious microbe drug sensitivity in blood, which comprises the following steps:
and (3) thallus enrichment: mixing the blood positive sample with a surfactant solution, centrifuging and removing a supernatant; mixing the obtained precipitate with a cleaning solution, centrifuging, and removing a supernatant to obtain precipitated thalli;
and (3) drug sensitivity detection: preparing a bacterial suspension; adding the bacterial suspension to a drug sensitive chip target plate, enabling the quantity of bacteria on each sample application position on the drug sensitive chip target plate to be lower than a mass spectrum detection threshold value, then carrying out incubation, absorbing and removing upper liquid after the incubation is finished, and carrying out mass spectrum detection on substances on the sample application positions on the drug sensitive chip target plate; and (3) judging the drug sensitive result according to the mass spectrum detection result, considering that the microorganism grows at the position where the microorganism is detected on the mass spectrum of the drug sensitive chip target plate, and considering that the microorganism growth is inhibited at the point where the microorganism cannot be detected, obtaining an MIC value according to the information, and determining sensitive, intermediary and drug resistant information according to CLSI, expert consensus and EUCAST guidelines.
Preferably, the drug sensitive chip target plate comprises a positive drug sensitive chip target plate and a negative drug sensitive chip target plate, the positive drug sensitive chip target plate is coated with antibiotic drugs suitable for positive bacteria, and the negative drug sensitive chip target plate is coated with antibiotic drugs suitable for negative bacteria;
before the drug sensitivity detection step, a blood gram staining step is also included, and the bacteria are determined to be positive bacteria or negative bacteria according to the gram staining step;
the drug sensitivity detection step comprises: selecting different drug sensitive chip target plates according to different strains:
when the bacteria are positive bacteria, selecting a positive bacteria drug sensitive chip target plate for drug sensitive detection;
when the bacteria are negative bacteria, the negative bacteria drug sensitive chip target plate is selected for drug sensitive detection.
Preferably, the positive drug sensitive chip target plate comprises a 96-hole plane target plate commonly used for mass spectrometry and antibiotics coated on the plane target plate, and the antibiotics and the main concentrations of the antibiotics are respectively added into 96 holes on the 96-hole plane target plate:
hole sites 1-4: amikacin (AN): 4,16, 32, 64
Hole site 5-9: gentamicin (GM): 1,2,4,8, 16
Hole sites 10-16: levofloxacin (LEV): 0.125,0.25,0.5,1,2,4,8
Hole sites 17-19: aztreonam (ATM): 4,8, 16
Hole sites 20-25: imipenem (IPM): 0.5,1,2,4,8, 16
Hole sites 26-31: meropenem (MEM): 0.25,0.5,1,2,4,8
Hole sites 32-37: tigecycline (TGC): 0.5,1,2,4,8, 16
Hole sites 38-41: cefuroxime (CXM): 1,4,8, 16
Hole sites 42-47: ceftazidime (CAZ): 1,2,4,8, 16, 32
Hole sites 48-54: ceftriaxone (CRO): 1,2,4,8, 16, 32, 64
Hole sites 55-60: cefepime (FEP): 1,2,4,8, 16, 32,
hole sites 61-64: cefoxitin (FOX): 8,16, 32, 64
Hole sites 65-69: polymyxin b (pb): 0.5,1,2,4,8
Hole sites 70-73: minocycline (MI): 2,4,8, 16
Hole sites 74-76: ampicillin/Sulbactam (SAM): 8/4, 16/8, 32/16
Hole sites 77-81: compound Sulfamethoxazole (SXT): 1/19,2/38,4/76,8/152, 16/304
Hole sites 82-85: ceftazidime/avibactam (CZA): 0.5/4,8/4, 16/4, 32/4
Hole sites 86-91: piperacillin/Tazobactam (TZP): 4/4,8/4, 16/4, 32/4, 64/4, 128/4
Hole 95: negative control
Well 96: and (4) positive control.
Preferably, the negative drug sensitive chip target plate comprises a 96-hole plane target plate commonly used for mass spectrometry and antibiotics coated on the plane target plate, and the antibiotics and the main concentrations of the antibiotics are respectively added into 96 holes on the 96-hole plane target plate:
hole sites 1-4: ciprofloxacin (CIP): 0.5,1,2,4,
hole sites are 5-8: tigecycline (TGC): 0.12,0.25,0.5,1
Hole sites 9-12: ampicillin (AM): 2,3,8, 16
Hole sites 13-16: rifampin (RA): 0.5,1,2,4,
hole site 17-20: cefoxitin (FOX): 2,4,8, 16,
hole sites 21-24: linezolid (LNZ): 1,2,4,8
Hole sites 25-29: clindamycin (CC): 0.25,0.5,1,2,4
Hole sites 30-34: gentamicin (GM): 1,2,4,8, 16
Hole sites 35-40: erythromycin (E): 0.25,0.5,1,2,4,8
Hole sites 41-46: minocycline (MI): 0.5,1,2,4,8, 16
Hole sites 47-50: compound Sulfamethoxazole (SXT): 0.5/9.5,1/19,2/38,4/76
Hole sites 51-56: oxacillin (OXA): 0.25,0.5,1,2,4,8
Hole sites 57-62: teicoplanin (TEC): 1,2,4,8, 16, 32
Hole sites 63-67: moxifloxacin (MXF): 0.125,0.25,0.5,1,2
Hole sites 68-75: penicillin (P): 0.125,0.25,0.5,1,2,4,8, 16
Hole sites 76-80: levofloxacin (LEV): 0.5,1,2,4,8
Hole sites 81-88: vancomycin (VA): 0.25,0.5,1,2,4,8, 16, 32
Hole 95: negative control
Well 96: and (4) positive control.
Preferably, the method further comprises a strain detection step before the drug sensitivity detection, wherein the strain detection comprises the following steps: and taking part of the thalli obtained in the step of thalli enrichment to perform strain identification to obtain the bacterial name.
Preferably, mass spectrometry is used for species identification.
Preferably, in the drug sensitivity detection step, the preparation of the bacterial suspension comprises:
when the bacteria are A-type bacteria, diluting the A-type bacteria by using a diluent to prepare a bacterial suspension with 0.5 McLeod turbidity, and then diluting by using a microbial liquid culture medium by 100-fold and 200-fold; wherein, the A-type bacteria are Enterobacteriaceae;
when the bacteria are B-type bacteria or C-type bacteria, diluting the bacteria with a diluent to prepare a bacterial suspension with 0.5 McLeod turbidity, and then diluting the bacterial suspension with a microbial liquid culture medium by 0.5 times of that of the A-type bacteria; wherein the B bacteria are Staphylococcus and enterococcus; the C-type bacteria are of the genus of monad and acinetobacter;
preferably, the diluent is normal saline;
preferably, the microbial broth is MH broth or BHI broth.
Preferably, the drug sensitive chip target plate comprises a positive drug sensitive chip target plate and a negative drug sensitive chip target plate, the positive drug sensitive chip target plate is coated with antibiotic drugs suitable for positive bacteria, and the negative drug sensitive chip target plate is coated with antibiotic drugs suitable for negative bacteria;
when the bacteria are A or C bacteria, selecting a negative bacteria drug sensitive chip target plate for drug sensitive detection;
when the bacteria is B type bacteria, a positive bacteria drug sensitive chip target plate is selected for drug sensitive detection.
Preferably, in the step of thallus enrichment, the surfactant is saponin, and the mass percentage concentration of the saponin solution is 2.5-8%; the volume ratio of the blood positive sample to the surfactant solution is (0.1-0.5): 1.
Preferably, the step of enriching the thallus comprises the following steps:
mixing the blood positive sample with a saponin solution, centrifuging for 3-12 min at 2500-2700 rpm, and removing the supernatant; mixing the obtained precipitate with normal saline, centrifuging for not less than 1min, and removing supernatant; mixing the obtained precipitate with normal saline, centrifuging for not less than 1min, and discarding supernatant to obtain thallus.
Preferably, in the step of cell enrichment,
the A-type bacteria comprise: escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae; the B-type bacteria comprise: staphylococcus aureus, Staphylococcus hominis, Staphylococcus capitis, Staphylococcus haemolyticus, enterococcus faecalis, enterococcus faecium
The class C bacteria include: acinetobacter baumannii and pseudomonas aeruginosa.
The invention provides a method for rapidly detecting infectious microbe drug sensitivity in blood. The method comprises the following steps: enriching thalli: mixing the blood positive sample with a surfactant solution, centrifuging and removing a supernatant; mixing the obtained precipitate with a cleaning solution, centrifuging, and removing a supernatant to obtain precipitated thalli; secondly, drug sensitivity detection: preparing a bacterial suspension; adding the bacterial suspension to a drug sensitive chip target plate, enabling the quantity of bacteria on each sample application position on the drug sensitive chip target plate to be lower than a mass spectrum detection threshold value, then carrying out incubation, absorbing and removing upper liquid after the incubation is finished, and carrying out mass spectrum detection on substances on the sample application positions on the drug sensitive chip target plate; and (3) judging the drug sensitive result according to the mass spectrum detection result, considering that the microorganism grows at the position where the microorganism is detected on the mass spectrum of the drug sensitive chip target plate, and considering that the microorganism growth is inhibited at the point where the microorganism cannot be detected, obtaining an MIC value according to the information, and determining sensitive, intermediary and drug resistant information according to CLSI, expert consensus and EUCAST guidelines. The invention has the technical effects that:
the invention can directly carry out the drug sensitivity test on the pretreated clinical yang blood reporting bottle without switching and purifying operations, and compared with the prior method, the invention can save time (at least 24h), has relatively simple and quick operation, does not need high manual ability, and has lower requirement on the professional degree of personnel. The saved precious time is used for the clinician to reasonably and accurately take medicine, the occurrence of drug-resistant bacteria is reduced, and the life of the patient is saved to the greatest extent.
Compared with the existing technology for detecting drug-resistant bacteria by mass spectrometry, the method can obtain the MIC value of the bacteria to various drugs, does not depend on any drug-resistant mechanism, can select the types and concentration gradients of the antibacterial drugs according to requirements, and has wide application range, rather than only judging whether the bacteria are drug-resistant.
The invention is based on a mass spectrum platform, the identification result and the drug sensitivity result can be synchronously output, and a bacterial protein fingerprint map can be obtained.
The method coats antibiotics with different types and concentrations on a sample target of a mass spectrometer, samples a sample to be detected at blank points of the sample target of the mass spectrometer, thereby realizing one-time identification, identifying the type of the microorganism and simultaneously identifying the MIC value of the microorganism to various antibiotics, and the whole identification process has simple operation flow and low dependence on operators and only needs 2-5 hours. Has important guiding significance for clinical treatment and drug selection dosage of bloodstream infectious diseases, especially critical patients.
Meanwhile, the diameter of a point position on the universal target plate for mass spectrometry identification is only 2.5mm, the area is small, too much liquid can be caused when bacterial suspension and antibiotic solution are dripped, the point position of a sample is too small to carry the too much liquid, the liquid of the sample point can overflow due to slight shaking or transfer, and the adjacent sample points are cross-polluted, so that the test fails; the sample plate is a plane, and after the antibiotic solution is dripped, the bacterial suspension cannot be blown and uniformly mixed, so that the sample plate has a large volume and is not thoroughly mixed, thalli cannot be fully contacted with the antibiotic solution, and false positive results (namely, the bacteria can be actually inhibited by the medicament, but the bacteria can grow due to insufficient contact) can be generated. The invention embeds and dries the antibiotic on the drug sensitive chip, after dropping the bacterial suspension, the dried antibiotic can be dissolved in the bacterial suspension, and the bacterial colony is deposited to the point position surface of the drug sensitive chip and fully contacts with the antibiotic, thereby the sample application is simple and convenient, and the drug sensitive detection accuracy is greatly improved.
Detailed Description
The invention discloses a method for rapidly detecting the susceptibility of infectious microorganisms in blood, which can be realized by appropriately improving process parameters by referring to the contents in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
MALDI-TOF MS is taken as a novel soft ionization mass spectrometry technology, and the taxonomic genus position of the unknown microorganism is determined mainly by collecting a protein fingerprint of the unknown microorganism and comparing the protein fingerprint with a protein map in a known database.
Adding surface active agents such as saponin and the like into a blood bottle reporting positive clinically, centrifuging, enriching thalli, selecting a part of thalli to be smeared on a blank sample plate, identifying and determining strains, selecting a proper antibiotic sample plate, selecting the antibiotic type required by drug sensitivity and the concentration range required to be prepared according to a mass spectrum identification result after identification is finished, embedding the prepared antibiotic on a mass spectrometer sample target plate, drying, re-suspending the enriched thalli by using physiological saline, preparing bacterial suspension with certain turbidity, diluting by using broth, enabling the diluted concentration to be smaller than a mass spectrum detection threshold value, spotting the diluted bacterial suspension on the sample plate coated with the antibiotic, incubating, discarding the broth after the incubation is finished, adding a microbial sample pretreatment reagent, and performing microbial drug resistance detection by using a MALDI TOF MS technology after drying, the growth point microbial protein reaches the mass spectrum detection threshold value, can be identified as drug resistance, inhibits the growth point microbial protein from being lower than the mass spectrum detection threshold value, has no identification result, and is sensitive, so that the drug sensitivity result and the accurate MIC value of the microorganism are obtained.
The invention directly omits the step of carrying out transfer culture on the blood culture positive specimen until the blood culture positive specimen grows out a single bacterial colony, thereby saving 5-18h for clinic; the method is relatively simple to operate, and most importantly, the method greatly shortens the time for identifying blood infectious microorganisms and detecting drug sensitivity, can quickly and accurately give identification and drug sensitivity results, and provides certain reference for clinical timely guidance of reasonable drug administration.
Preferably, the microorganism broth is MH broth or BHI broth.
Preferably, the microbial liquid medium is MH liquid medium.
In the present invention, the antibiotic on the target plate of the drug sensitive chip is selected from, but not limited to, amikacin, gentamicin, levofloxacin, aztreonam, imipenem, meropenem, tigecycline, cefuroxime, ceftazidime, ceftriaxone, cefepime, cefoxitin, polymyxin B, minocycline, ampicillin sulbactam, fantail-nimine, ceftazidime abatan, piperacillin tazobactam, cefoperazone sulbactam, ciprofloxacin, tigecycline, ampicillin, rifampin, cefoxitin, linezolid, clindamycin, gentamicin, erythromycin, minocycline, porfuginine, fenzamide, teicoplanin, moxifloxacin, penicillin, levofloxacin, vancomycin.
The invention provides a method for quickly identifying and quickly testing drug sensitivity of microorganisms in blood stream infection, which can separate out pure microorganisms through quick pretreatment after a blood culture bottle reports positive, the obtained thalli is resuspended by normal saline and prepared into bacterial suspension with certain turbidity, the bacterial suspension is diluted by broth and is spotted on plates coated with antibiotics with different types and concentrations, after a period of incubation, mass spectrum is used for detection, the principle that mass spectrum can be used for identifying through detecting microbial ribosomal protein is utilized, microbial protein exists at a growth point, identification can be carried out, the growth point can be inhibited from being detected with mycoprotein, no identification result exists, and thus the drug sensitivity result and the accurate MIC value of the microorganisms are obtained.
The invention can break through the inherent mode that the blood culture positive specimen can be subjected to drug sensitive detection after being transferred to the solid culture medium for culture, can break through the bottleneck that the culture time of bacteria/fungi is long, has short detection time and simple operation steps, can obtain an accurate MIC value, provides a certain reference for clinical timely and rapid medication, and strives for valuable treatment time for patients with clinical severe bloodstream infection.
The reagents or apparatus used in the present invention are commercially available.
The invention is further illustrated by the following examples:
example 1
The invention provides a method for rapidly detecting infectious microbe drug sensitivity in blood, which mainly comprises the following steps:
(1) enrichment of thallus
Extracting a blood sample from a yang-reporting blood bottle, adding a surfactant, and uniformly mixing; and then centrifuging for multiple times at different centrifugation speeds, sucking and removing supernatant after each centrifugation, and adding physiological saline to continue the centrifugation until the centrifugation is finished. The surfactant is used for enriching the broken blood cell thallus, and can adopt 2.5-8% of saponin. The specific operation is as follows:
firstly, 1mL of blood is extracted from a yang-reporting blood bottle into a 1.5mL of EP tube; adding 4% saponin solution at a ratio of 0.2:1 (adding 0.2 unit volume of saponin solution into unit volume of blood sample), and mixing;
second, centrifuge tube 2600rpm for 5min and discard supernatant. Adding 1mL of normal saline, blowing, beating and uniformly mixing; 13000rmp for 2min, discarding the supernatant; the obtained precipitate was mixed with physiological saline, centrifuged at 13000rpm for 2min, and the supernatant was discarded to obtain the cell precipitated at the bottom of the centrifuge tube.
(2) Drug susceptibility testing
Firstly, preparing bacterial suspension, and enabling the quantity of thalli on each sample application position on a target plate of a drug sensitive chip to be lower than a mass spectrum detection threshold value, namely, after the diluted thalli are dripped on the target plate according to the volume of a solution subjected to normal mass spectrum sample application, the quantity of the thalli is lower than the mass spectrum detection threshold value, and the thalli cannot be detected due to too small quantity of the thalli when the bacterial suspension is directly subjected to mass spectrum detection. The specific operation is as follows: the enriched bacteria were prepared into a bacterial suspension with 0.5 McLeod's turbidity using physiological saline, and then diluted 100-fold with MH liquid medium (also called MH nutrient broth).
Secondly, preparing a drug sensitive chip target plate, comprising: antibiotic selection: according to the difference of negative bacteria, positive bacteria and fungi in different clinical specimens such as blood specimens, urine specimens, lavage fluid specimens, sputum specimens and the like and the difference of antibacterial spectrum of different medicines, suitable antibiotics are selected, Amikacin (AN), Gentamicin (GM), Levofloxacin (LEV), Aztreonam (ATM), imipenem, meropenem, tigecycline, cefuroxime, ceftazidime, ceftriaxone, cefepime, cefoxitin, polymyxin B, minocycline, ampicillin sulbactam, compound sulfamethoxazole, ceftazidime, piperacillin tazobactam and cefoperazone sulbactam are selected as medicines used as a positive drug sensitive plate; ciprofloxacin, tigecycline, ampicillin, rifampicin, cefoxitin, linezolid, clindamycin, gentamicin, erythromycin, minocycline, porfuzone sulfamethoxazole, oxacillin, teicoplanin, moxifloxacin, penicillin, levofloxacin and vancomycin are selected as the medicines of the negative drug sensitive plate.
Preparing an antibiotic solution: adding antibiotics into a solvent to prepare antibiotic solutions with different concentrations, wherein the antibiotic solutions specifically comprise the concentrations of the hole sites on the negative drug sensitive plate and the positive drug sensitive plate, and the sequence of the hole sites can be interchanged; the specific operation is that different solvents such as water, DMSO, ethanol solution, propanesulfonic acid solution, ethanesulfonic acid solution and the like are selected according to different antibiotic types, and the pH value is adjusted to be proper according to the antibiotic properties; an antibiotic protective agent is added when the antibiotic solution is prepared, so that the antibiotic is protected and the antibiotic is prevented from losing efficacy in the shelf life; the antibiotic protective agent can adopt organosilicon surfactant, cilastatin sodium and the like.
③ coating antibiotics: the prepared antibiotic solution is coated on a chip by a coating machine or a pipette, the chip can be a metal plate, a silicon plate, a plastic plate, a paper plate and the like, and the preferred chip in the embodiment is a target plate used for mass spectrometry. Then selecting a proper drying method for drying according to the characteristics of the antibiotics, wherein the drying method comprises the following steps of (1) heating and air-blast drying; (2) low-temperature vacuum drying; (3) low-temperature adsorption drying; (4) drying at normal temperature and low humidity, and performing light-proof treatment on some photosensitive antibiotics or antibiotics which are easily degraded at high temperature, or selecting a low-temperature drying mode, wherein the drying time is 2-48 h.
And fourthly, preservation: and (4) putting the dried chip into a packaging box, and carrying out vacuum light-resistant treatment for later use.
Thirdly, dropping the bacterial suspension, and detecting after incubation
Adding the bacterial suspension to a drug sensitive chip target plate, wherein the dropping volume of the bacterial suspension is the same as the antibiotic labeling volume on the drug sensitive chip target plate, the volume of the bacterial suspension is preferably 3 mu l, then placing the bacterial suspension in an environment with constant temperature of 37 ℃ and relative humidity of 99.9% for incubation for 4 hours, absorbing the upper liquid after the incubation is finished, treating the substance on the sample spotting position on the drug sensitive chip target plate according to a mass spectrum detection pretreatment method, and detecting the substance on a mass spectrometer. The specific operation is as follows: after incubation is completed, an absorbing and discarding device is used for discarding redundant culture medium on the drug sensitive chip, because thallus can be precipitated to the bottom of the drug sensitive chip in the growth process, only liquid above the drug sensitive chip is absorbed and discarded when the culture medium is absorbed and discarded, the thallus can not be taken away, the drug sensitive chip and the thallus and participating culture medium above the drug sensitive chip are dried after the culture medium is absorbed and discarded, a pretreatment reagent is dripped after drying, and a mass spectrometer is used for detecting after the pretreatment reagent is dried. The excess liquid nutrient medium is sucked and discarded in the step, so that the mass nutrient components in the nutrient medium are prevented from influencing the mass spectrum identification result after the nutrient medium is directly dried, and after most of broth is sucked and discarded, the trace broth remained on the surface of the drug sensitive chip contains fewer nutrient components, so that the accuracy of the mass spectrum identification result is not influenced.
Finally, the drug sensitive result is interpreted according to the mass spectrum detection result, the sample application position of the microorganism is identified by the mass spectrum on the drug sensitive chip target plate to judge drug resistance (namely, the microorganism is insensitive to the antibiotic with corresponding concentration on the sample application position, resists the drug and continues to grow, the number of the microorganism is increased, so the microorganism can be detected by the mass spectrometer), and the position of the microorganism detected by the mass spectrum on the drug sensitive chip target plate is considered to haveThe microorganism growth is considered to be inhibited at the position where the microorganism growth is not detected, the number of the microorganisms is maintained as it is or the growth is extremely slow, and the number of the microorganisms hardly reaches the mass spectrum detection threshold (the mass spectrum detection threshold is 10) within a fixed incubation time4-106CFU/ml), so the mass spectrometer does not detect the bacteria. Obtaining MIC value according to the information, and determining sensitive, intermediary and drug resistance information according to the guidelines of CLSI, expert consensus, EUCAST and the like.
In the embodiment, a thallus enrichment method is adopted to quickly enrich thallus in a positive blood bottle, thallus is prepared into a thallus suspension and is dripped onto a drug sensitive chip target plate for incubation detection, and the growth condition of the thallus is detected by utilizing the advantage of high detection sensitivity of a mass spectrometer, so that the drug sensitive detection time is greatly reduced, and accurate guidance is provided for clinical medication of clinical critically ill patients.
Example 2
The invention provides a method for rapidly detecting infectious microbe drug sensitivity in blood, which mainly comprises the following steps:
(1) enrichment of thallus
Extracting a blood sample from a yang-reporting blood bottle, adding a surfactant, and uniformly mixing; and then centrifuging for multiple times at different centrifugation speeds, sucking and removing supernatant after each centrifugation, and adding physiological saline to continue the centrifugation until the centrifugation is finished. The surfactant is used for enriching the broken blood cell thallus, and can adopt 2.5-8% of saponin. The specific operation is as follows:
firstly, 1mL of blood is extracted from a yang-reporting blood bottle into a 1.5mL of EP tube; adding 4% saponin solution at a ratio of 0.2:1 (adding 0.2 unit volume of saponin solution into unit volume of blood sample), and mixing;
second, centrifuge tube 2600rpm for 5min and discard supernatant. Adding 1mL of normal saline, blowing, beating and uniformly mixing; 13000rmp for 2min, discarding the supernatant; the obtained precipitate was mixed with physiological saline, centrifuged at 13000rpm for 2min, and the supernatant was discarded to obtain the cell precipitated at the bottom of the centrifuge tube.
(2) Gram stain determination
Gram staining is carried out on a blood sample in the yang blood reporting bottle, and the bacteria are judged to be positive bacteria or negative bacteria.
(3) Drug susceptibility testing
Preparing a bacterial suspension:
the specific operation is as follows: and (3) preparing the enriched thalli into bacterial suspension with the turbidity of 0.5 McLeod by using normal saline, and diluting by 100 times by using an MH liquid culture medium.
Sample application incubation detection:
in this embodiment, the drug sensitive chip target plate comprises a positive drug sensitive chip target plate and a negative drug sensitive chip target plate, the positive drug sensitive chip target plate is coated with antibiotic drugs suitable for positive bacteria, the positive drug sensitive chip target plate comprises a 96-hole plane target plate commonly used for mass spectrometry and antibiotic drugs coated on the plane target plate, and the antibiotic and the main concentration added in 96 holes on the 96-hole plane target plate are respectively:
hole sites 1-4: amikacin (AN): 4,16, 32, 64
Hole site 5-9: gentamicin (GM): 1,2,4,8, 16
Hole sites 10-16: levofloxacin (LEV): 0.125,0.25,0.5,1,2,4,8
Hole sites 17-19: aztreonam (ATM): 4,8, 16
Hole sites 20-25: imipenem: 0.5,1,2,4,8, 16
Hole sites 26-31: meropenem: 0.25,0.5,1,2,4,8
Hole sites 32-37: tigecycline: 0.5,1,2,4,8, 16
Hole sites 38-41: cefuroxime: 1,4,8, 16
Hole sites 42-47: ceftazidime: 1,2,4,8, 16, 32
Hole sites 48-54: ceftriaxone: 1,2,4,8, 16, 32, 64
Hole sites 55-60: cefepime: 1,2,4,8, 16, 32,
hole sites 61-64: cefoxitin: 8,16, 32, 64
Hole sites 65-69: polymyxin B: 0.5,1,2,4,8
Hole sites 70-73: minocycline: 2,4,8, 16
Hole sites 74-76: ampicillin/sulbactam: 8/4, 16/8, 32/16
Hole sites 77-81: compound sulfamethoxazole: 1/19,2/38,4/76,8/152, 16/304
Hole sites 82-85: ceftazidime/avibactam: 0.5/4,8/4, 16/4, 32/4
Hole sites 86-91: piperacillin/tazobactam: 4/4,8/4, 16/4, 32/4, 64/4, 128/4.
Antibiotic medicine suitable for negative bacteria is coated on the negative medicine sensitive chip target plate; the negative drug sensitive chip target plate comprises a 96-hole plane target plate commonly used for mass spectrometry and antibiotics coated on the plane target plate, wherein the antibiotics and the main concentrations of the antibiotics are respectively added into 96 holes on the 96-hole plane target plate:
hole sites 1-4: ciprofloxacin: 0.5,1,2,4,
hole sites are 5-8: tigecycline: 0.12,0.25,0.5,1
Hole sites 9-12: ampicillin: 2,3,8, 16
Hole sites 13-16: rifampicin: 0.5,1,2,4,
hole site 17-20: cefoxitin: 2,4,8, 16,
hole sites 21-24: linezolid: 1,2,4,8
Hole sites 25-29: clindamycin: 0.25,0.5,1,2,4
Hole sites 30-34: gentamicin: 1,2,4,8, 16
Hole sites 35-40: erythromycin: 0.25,0.5,1,2,4,8
Hole sites 41-46: minocycline: 0.5,1,2,4,8, 16
Hole sites 47-50: compound sulfamethoxazole: 0.5/9.5,1/19,2/38,4/76
Hole sites 51-56: oxacillin: 0.25,0.5,1,2,4,8
Hole sites 57-62: teicoplanin: 1,2,4,8, 16, 32
Hole sites 63-67: moxifloxacin: 0.125,0.25,0.5,1,2
Hole sites 68-75: penicillin: 0.125,0.25,0.5,1,2,4,8, 16
Hole sites 76-80: levofloxacin: 0.5,1,2,4,8
Hole sites 81-88: vancomycin: 0.25,0.5,1,2,4,8, 16, 32.
According to the result of gram staining judgment, selecting a proper drug sensitive chip target plate (specifically, a positive bacterial suspension is dripped to the positive drug sensitive chip target plate, a negative bacterial suspension is dripped to the negative drug sensitive chip target plate), adding the bacterial suspension onto the drug sensitive chip target plate, wherein the dripping volume of the bacterial suspension is the same as the antibiotic labeling volume on the drug sensitive chip target plate, the volume of the bacterial suspension is preferably 3 mu l, then placing the bacterial suspension in an environment with constant temperature of 37 ℃ and relative humidity of 99.9 percent for incubation for 4 hours, sucking and discarding the upper liquid after the incubation is finished, and performing mass spectrum detection on substances on the sample spotting position on the drug sensitive chip target plate.
And (3) judging the drug sensitivity result according to the mass spectrum detection result, wherein the judgment of the drug sensitivity result in the embodiment is the same as that in the embodiment 1, and the details are not repeated.
In the embodiment, the gram staining step is added to quickly judge whether the bacteria are positive bacteria or negative bacteria, so that the drug sensitive chip target plate can be selected in a targeted manner, and the waste of the antibiotic drug sensitive chip target plate is avoided.
Example 3
The invention provides a method for rapidly detecting infectious microbe drug sensitivity in blood, which mainly comprises the following steps:
(1) enrichment of thallus
Extracting a blood sample from a yang-reporting blood bottle, adding a surfactant, and uniformly mixing; and then centrifuging for multiple times at different centrifugation speeds, sucking and removing supernatant after each centrifugation, and adding physiological saline to continue the centrifugation until the centrifugation is finished. The surfactant is used for enriching the broken blood cell thallus, and can adopt 2.5-8% of saponin. The specific operation is as follows:
firstly, 1mL of blood is extracted from a yang-reporting blood bottle into a 1.5mL of EP tube; adding 6% saponin solution at a ratio of 0.2:1 (adding 0.2 unit volume of saponin solution into unit volume of blood sample), and mixing;
second, centrifuge tube 2600rpm for 5min and discard supernatant. Adding 1mL of normal saline, blowing, beating and uniformly mixing; 13000rmp for 2min, discarding the supernatant; the obtained precipitate was mixed with physiological saline, centrifuged at 13000rpm for 2min, and the supernatant was discarded to obtain the cell precipitated at the bottom of the centrifuge tube.
(2) Strain detection
Specifically, the enriched bacteria are smeared on a sample plate, a pretreatment reagent is added, drying is carried out, mass spectrum identification is carried out, and the name of the bacteria is determined; at the moment, the thalli contains trace impurities, the morphology of the aseptic body cannot be subjected to morphological observation to determine the bacterial name, mass spectrometry identification has no requirement on the morphology of the bacteria, and the small amount of impurities and protein molecules of the bacteria are not at a molecular weight level, so that mass spectrometry identification cannot be influenced, the bacterial name can be quickly identified by adopting mass spectrometry, and the conventional culture and purification time is saved. The whole strain identification process can be completed in about ten minutes.
(3) Drug susceptibility testing
Preparing a bacterial suspension:
according to the detection result, dividing the bacteria into A-type bacteria, B-type bacteria and C-type bacteria; wherein, the A-type bacteria are Enterobacteriaceae; the B bacteria are Staphylococcus and enterococcus; the C-type bacteria are of the genus Pseudomonas or Acinetobacter.
When the bacteria are A bacteria, diluting the A bacteria with normal saline to prepare bacterial suspension with 0.5 McLeod turbidity, and diluting 200 times with MH liquid culture medium for later use.
When the bacteria are B bacteria or C bacteria, diluting with normal saline to obtain bacterial suspension with turbidity of 0.5 McLeod, and diluting with MH liquid culture medium by 100 times.
Sample application incubation detection:
in this embodiment, the drug sensitive chip target plate includes a positive drug sensitive chip target plate and a negative drug sensitive chip target plate, and the information of the negative drug sensitive chip target plate and the positive drug sensitive chip target plate are the same as those in embodiment 2, and are not described herein again.
According to the result of strain detection, when the bacteria are A or C type bacteria, selecting a negative bacteria drug sensitive chip target plate for drug sensitive detection; when the bacteria are B-type bacteria, a positive bacteria drug sensitive chip target plate is selected for drug sensitive detection, and the method specifically comprises the following steps: adding the bacterial suspension onto the drug sensitive chip target plate, wherein the dropping volume of the bacterial suspension is the same as the antibiotic labeling volume on the drug sensitive chip target plate, the volume of the bacterial suspension is preferably 3 mu l, then placing the bacterial suspension in an environment with constant temperature of 37 ℃ and relative humidity of 99.9% for incubation, and incubating for 3 hours when the bacteria are type A bacteria and incubating for 4 hours when the bacteria are type B or type C bacteria. In this embodiment, the class a bacteria include: escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae; the B-type bacteria comprise: staphylococcus hominis, staphylococcus capitis, staphylococcus haemolyticus, enterococcus faecalis, enterococcus faecium; the class C bacteria comprise: acinetobacter baumannii and pseudomonas aeruginosa.
Since the growth cycles of the B-type bacteria and the C-type bacteria are slower than those of the A-type bacteria, and the protein sensitivity (namely mass spectrum detection threshold and thallus density) detectable by a mass spectrometer is basically the same, the dilution times of the B-type bacteria and the C-type bacteria are required to be reduced, so that the density of the B-type bacteria and the C-type bacteria is equivalent to that of the A-type bacteria, and the incubation time is reduced.
After the incubation is finished, the upper liquid is sucked away, and mass spectrum detection is carried out on the substance on the sample spotting position on the drug sensitive chip target plate.
And (3) judging the drug sensitivity result according to the mass spectrum detection result, and judging the drug sensitivity result according to the mass spectrum detection result, wherein the judgment of the drug sensitivity result in the embodiment is the same as that in the embodiment 1, and is not repeated herein.
In the embodiment, the bacterial name is rapidly judged by adding the bacterial detection step, so that a proper dilution multiple is selected according to the form and growth habit of the bacteria, the incubation time is shortened as far as possible, the drug sensitive detection time is shortened, the targeted drug sensitive chip target plate is conveniently selected in a targeted manner, and the waste of the antibiotic drug sensitive chip target plate is avoided.
All examples of the present invention were mass spectrometric identification using an Autofms1000 mass spectrometer manufactured by Zhengzhou Antu laboratory instruments Ltd.
Test example 1 examination of saponin concentration
Taking 10 parts of clinical yang blood reporting bottles with the serial numbers of 1, 2, 3 and … … 10 respectively; taking 6mL of blood from each blood bottle, dividing equally, loading into EP tube with number A, B, C, D, E, F, adding 0.2mL of saponin solution with concentration of 1.5%, 2.5%, 3.5%, 5%, 6%, 7%, respectively, and mixing well, wherein the saponin is 10-25% pure product of Sigma company. Then centrifuging the samples at 2600rpm for 5min respectively, discarding the supernatant, adding 1mL of physiological saline, and uniformly blowing; 13000rmp for 2min, discarding the supernatant; this operation was repeated once to obtain the cells precipitated at the bottom of the centrifuge tube. Respectively carrying out mass spectrum pretreatment on the obtained thalli, and then carrying out strain detection by using a time-of-flight mass spectrometer, wherein the detection results are as follows:
TABLE 1
Remarking: description of the identification score:
[9.5, 10.0]: species level confidence, possible subspecies;
[9.0,9.5): seed level confidence;
[6.0,9.0): an attribute level confidence;
[0.0,6.0): is not trusted.
In the experiment, a mass spectrometer of model Autofms1000 manufactured by Anji laboratory instruments (Zhengzhou) is adopted for strain detection. The mass spectrum identification score represents identification accuracy, the higher the score is, the higher the identification accuracy is, and when the identification score is lower than a certain threshold value, the identification result is not credible.
From the detection results, the identification accuracy is highest when the concentration of the saponin is 2.5% -7%, and is lower when the concentration is 1.5% or 2.5%, because the saponin is a surfactant and mainly has the functions of cracking blood cells, white blood cells, platelets and the like, and when the concentration of the saponin is lower, the added saponin is not enough to crack target cells, so that the precipitate obtained by the next step of centrifugation also contains part of impurity cells, and the identification accuracy is influenced.
Test example 2
And (3) performing thallus enrichment by using another surfactant SDS solution instead of saponin:
taking 10 parts of clinical yang blood reporting bottles with the serial numbers of 1, 2, 3 and … … 10 respectively; 6mL of blood in each blood bottle was collected and divided into equal portions, and the equal portions were put into an EP tube No. A, B, C, D, E, F, and 0.2mL of SDS solutions of 5%, 10%, 15% and 20% concentration were added and mixed. Then centrifuging the samples at 2600rpm for 5min respectively, discarding the supernatant, adding 1mL of physiological saline, and uniformly blowing; 13000rmp for 2min, discarding the supernatant; adding 1mL of physiological saline into the obtained precipitate, and uniformly mixing by blowing; 13000rmp for 2min, and the supernatant was discarded to obtain the cells precipitated at the bottom of the centrifuge tube. Respectively carrying out mass spectrum pretreatment on the obtained thalli, and then carrying out strain detection by using a time-of-flight mass spectrometer, wherein the detection results are as follows:
TABLE 2
Test example 3
And (3) performing thallus enrichment by using another surfactant triton X-100 solution instead of saponin:
taking 10 parts of clinical yang blood reporting bottles with the serial numbers of 1, 2, 3 and … … 10 respectively; 6mL of blood from each blood bottle was divided into equal portions, and the equal portions were placed in an EP tube No. A, B, C, D, E, F, and 0.2mL of triton X-100 solutions with concentrations of 0.1%, 0.5%, 1.0%, and 1.5% were added and mixed. Then centrifuging the samples at 2600rpm for 5min respectively, discarding the supernatant, adding 1mL of physiological saline, and uniformly blowing; 13000rmp for 2min, discarding the supernatant; adding 1mL of physiological saline into the obtained precipitate, and uniformly mixing by blowing; 13000rmp for 2min, and the supernatant was discarded to obtain the cells precipitated at the bottom of the centrifuge tube. Respectively carrying out mass spectrum pretreatment on the obtained thalli, and then carrying out strain detection by using a time-of-flight mass spectrometer, wherein the detection results are as follows:
TABLE 3
From the above test examples 1 to 3, it is understood that the mass spectrometric identification score of the cells enriched with 2.5% to 7% saponin solution in the surfactant is higher than that of the cells enriched with SDS solution and triton X-100 solution, because the 2.5% to 7% saponin solution can better lyse blood cells, and the enriched cells are purer by washing and centrifugation.
Test example 4 investigation of dilution factor and dropping volume of bacterial suspension
On the basis of 3.5 percent of saponin concentration in example 1, taking Escherichia coli in negative bacteria as an example, referring to a conventional drug sensitive test method-broth dilution method of CLSI M100, and considering both a drug sensitive chip and a mass spectrum detection threshold value. The invention selects clinical yang blood bottle (n is 50), and uses normal saline to prepare 0.5 McLeod turbidity (about 1.5X 10)8CFU/mL) were diluted 50-fold, 100-fold, 150-fold, 200-fold and 300-fold respectively with MH broth in 5 gradients. The dropping volumes were set to 10 gradients of 1. mu.L, 2. mu.L, 3. mu.L, 4. mu.L, 5. mu.L, 6. mu.L, 7. mu.L, 8. mu.L, 9. mu.L, and 10. mu.L. Incubate at 37 ℃ for 4 h. The specific results are as follows:
TABLE 4
And a result judgment method comprises the following steps: according to the guidelines of CLSI, M52 and the like, each antibiotic CA and EA of each strain is more than 90%, and VME is less than 3%; ME < 3% was considered satisfactory. Wherein the meaning of each parameter is as follows:
(1) category Agreement (CA): the drug sensitivity results of the evaluation method and the reference method are consistent with the sensitivity, mediation, dose-dependent sensitivity and drug resistance judged according to the break points.
(2) The basal agreement (EA) assessment method measures bacterial MICs to within 1 log-fold dilution from the reference method.
(3) Small error (minor error, mE): the drug sensitivity results of the reference methods and the evaluation methods, one being an intermediary and the other being sensitive or resistant.
(4) Gross error (ME): the reference method is sensitive to the drug sensitivity result, and the evaluation method is drug resistance.
(5) Very significant error (verymajor error, VME): the reference method is drug-resistant, and the evaluation method is sensitive.
And (4) analyzing results: the test takes single drug CA as an evaluation index, and the lowest and highest combinations are respectively between 84% and 98%: the dilution factor is 300 times, the dropping volume is 10 mu L, and the dilution factor is 150 times, and the dropping volume is 9 mu L. The same or better than the current automated drug sensitive method and paper sheet method.
Test example 5 accuracy of drug sensitivity
With the optimal values, the drug sensitivity test is carried out according to 6 bacteria such as staphylococcus aureus, escherichia coli, klebsiella pneumoniae, enterococcus faecium, pseudomonas aeruginosa, staphylococcus epidermidis and the like which are common bacteria infected by clinical blood stream. The specific results are as follows:
TABLE 5 summary of positive bacteria evaluation results
TABLE 6 summary of the negative bacteria susceptibility test results
The accurate evaluation method is the same as in test example 4, and is not described herein again.
The accuracy of each antibiotic in the drug sensitivity test of 6 common pathogenic bacteria for the current bloodstream infection is within the standard requirement.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for rapidly detecting infectious microbe drug sensitivity in blood is characterized by comprising the following steps:
and (3) thallus enrichment: mixing the blood positive sample with a surfactant solution, centrifuging and removing a supernatant; mixing the obtained precipitate with a cleaning solution, centrifuging, and removing a supernatant to obtain precipitated thalli;
and (3) drug sensitivity detection: preparing a bacterial suspension; adding the bacterial suspension to a drug sensitive chip target plate, enabling the quantity of bacteria on each sample application position on the drug sensitive chip target plate to be lower than a mass spectrum detection threshold value, then carrying out incubation, absorbing and removing upper liquid after the incubation is finished, and carrying out mass spectrum detection on substances on the sample application positions on the drug sensitive chip target plate; and (3) judging the drug sensitive result according to the mass spectrum detection result, considering that the microorganism grows at the position where the microorganism is detected on the mass spectrum of the drug sensitive chip target plate, and considering that the microorganism growth is inhibited at the point where the microorganism cannot be detected, obtaining an MIC value according to the information, and determining sensitive, intermediary and drug resistant information according to CLSI, expert consensus and EUCAST guidelines.
2. The method of claim 1,
the drug sensitive chip target plate comprises a positive drug sensitive chip target plate and a negative drug sensitive chip target plate, wherein the positive drug sensitive chip target plate is coated with antibiotic drugs suitable for positive bacteria, and the negative drug sensitive chip target plate is coated with antibiotic drugs suitable for negative bacteria;
the method also comprises a blood gram staining step before the drug sensitivity detection step, and the bacteria are determined to be positive bacteria or negative bacteria according to the gram staining step;
the drug sensitivity detection step comprises: selecting different drug sensitive chip target plates according to different strains:
when the bacteria are positive bacteria, selecting a positive bacteria drug sensitive chip target plate for drug sensitive detection;
when the bacteria are negative bacteria, the negative bacteria drug sensitive chip target plate is selected for drug sensitive detection.
3. The method of claim 2, wherein the positive drug sensitive chip target plate comprises a 96-well planar target plate and antibiotics coated on the planar target plate, and the antibiotics and the main concentrations are respectively added in 96-well sites on the 96-well planar target plate:
hole sites 1-4: amikacin (AN): 4,16, 32, 64
Hole site 5-9: gentamicin (GM): 1,2,4,8, 16
Hole sites 10-16: levofloxacin (LEV): 0.125,0.25,0.5,1,2,4,8
Hole sites 17-19: aztreonam (ATM): 4,8, 16
Hole sites 20-25: imipenem (IPM): 0.5,1,2,4,8, 16
Hole sites 26-31: meropenem (MEM): 0.25,0.5,1,2,4,8
Hole sites 32-37: tigecycline (TGC): 0.5,1,2,4,8, 16
Hole sites 38-41: cefuroxime (CXM): 1,4,8, 16
Hole sites 42-47: ceftazidime (CAZ): 1,2,4,8, 16, 32
Hole sites 48-54: ceftriaxone (CRO): 1,2,4,8, 16, 32, 64
Hole sites 55-60: cefepime (FEP): 1,2,4,8, 16, 32,
hole sites 61-64: cefoxitin (FOX): 8,16, 32, 64
Hole sites 65-69: polymyxin b (pb): 0.5,1,2,4,8
Hole sites 70-73: minocycline (MI): 2,4,8, 16
Hole sites 74-76: ampicillin/Sulbactam (SAM): 8/4, 16/8, 32/16
Hole sites 77-81: compound Sulfamethoxazole (SXT): 1/19,2/38,4/76,8/152, 16/304
Hole sites 82-85: ceftazidime/avibactam (CZA): 0.5/4,8/4, 16/4, 32/4
Hole sites 86-91: piperacillin/Tazobactam (TZP): 4/4,8/4, 16/4, 32/4, 64/4, 128/4
Hole 95: negative control
Well 96: and (4) positive control.
4. The method of claim 2, wherein the negative drug sensitive chip target plate comprises a 96-well planar target plate and antibiotics coated on the planar target plate, and the antibiotics and the main concentrations are respectively added in 96-well sites on the 96-well planar target plate:
hole sites 1-4: ciprofloxacin (CIP): 0.5,1,2,4,
hole sites are 5-8: tigecycline (TGC): 0.12,0.25,0.5,1
Hole sites 9-12: ampicillin (AM): 2,3,8, 16
Hole sites 13-16: rifampin (RA): 0.5,1,2,4,
hole site 17-20: cefoxitin (FOX): 2,4,8, 16,
hole sites 21-24: linezolid (LNZ): 1,2,4,8
Hole sites 25-29: clindamycin (CC): 0.25,0.5,1,2,4
Hole sites 30-34: gentamicin (GM): 1,2,4,8, 16
Hole sites 35-40: erythromycin (E): 0.25,0.5,1,2,4,8
Hole sites 41-46: minocycline (MI): 0.5,1,2,4,8, 16
Hole sites 47-50: compound Sulfamethoxazole (SXT): 0.5/9.5,1/19,2/38,4/76
Hole sites 51-56: oxacillin (OXA): 0.25,0.5,1,2,4,8
Hole sites 57-62: teicoplanin (TEC): 1,2,4,8, 16, 32
Hole sites 63-67: moxifloxacin (MXF): 0.125,0.25,0.5,1,2
Hole sites 68-75: penicillin (P): 0.125,0.25,0.5,1,2,4,8, 16
Hole sites 76-80: levofloxacin (LEV): 0.5,1,2,4,8
Hole sites 81-88: vancomycin (VA): 0.25,0.5,1,2,4,8, 16, 32
Hole 95: negative control
Well 96: and (4) positive control.
5. The method according to any one of claims 1 to 4, further comprising a strain detection step prior to the drug susceptibility detection, said strain detection comprising: and taking part of the thalli obtained in the thalli enrichment step for strain identification to obtain a bacterial name.
6. The method of claim 5, wherein in the susceptibility testing step, the preparation of the bacterial suspension comprises:
when the bacteria are A-type bacteria, diluting the A-type bacteria by using a diluent to prepare a bacterial suspension with 0.5 McLeod turbidity, and then diluting by using a microbial liquid culture medium by 100-fold and 200-fold; wherein, the A-type bacteria are Enterobacteriaceae;
when the bacteria are B-type bacteria or C-type bacteria, diluting the bacteria with a diluent to prepare a bacterial suspension with 0.5 McLeod turbidity, and then diluting the bacterial suspension with a microbial liquid culture medium by 0.5 times of that of the A-type bacteria; wherein the B bacteria are Staphylococcus and enterococcus; the C-type bacteria are of the genus Pseudomonas or Acinetobacter.
7. The method of claim 6,
the drug sensitive chip target plate comprises a positive drug sensitive chip target plate and a negative drug sensitive chip target plate, wherein the positive drug sensitive chip target plate is coated with antibiotic drugs suitable for positive bacteria, and the negative drug sensitive chip target plate is coated with antibiotic drugs suitable for negative bacteria;
when the bacteria are A or C bacteria, selecting a negative bacteria drug sensitive chip target plate for drug sensitive detection;
when the bacteria is B type bacteria, a positive bacteria drug sensitive chip target plate is selected for drug sensitive detection.
8. The method according to any one of claims 1 to 4, wherein in the step of thallus enrichment, the surfactant is saponin, and the mass percentage concentration of the saponin solution is 2.5-8%; the volume ratio of the blood positive sample to the surfactant solution is (0.1-0.5): 1.
9. The method according to claim 8, wherein the step of enriching the biomass comprises:
mixing the blood positive sample with a saponin solution, centrifuging for 3-12 min at 2500-2700 rpm, and removing the supernatant; mixing the obtained precipitate with normal saline, centrifuging for not less than 1min, and removing supernatant; mixing the obtained precipitate with normal saline, centrifuging for not less than 1min, and discarding supernatant to obtain thallus.
10. The method according to claim 6, wherein, in the step of cell enrichment,
the A-type bacteria comprise: escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae; the B-type bacteria comprise: staphylococcus aureus, staphylococcus hominis, staphylococcus capitis, staphylococcus haemolyticus, enterococcus faecalis, enterococcus faecium;
the class C bacteria comprise: acinetobacter baumannii and pseudomonas aeruginosa.
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